G protein-coupled receptors detect many different external signals including ones that regulate gene expression and cellular differentiation. While many components of G protein-mediated signal transduction pathways are known, the mechanisms by which different signals regulate specific genes remains to be determined. Pathway specificity is, in part, determined by the G protein G (alpha) subunits, and recent data indicate these subunits play a role in gene regulation in the soil amoebae Dictyostelium. The goal of the proposed research is to test the hypothesis that G (alpha) subunits regulate gene expression through interactions with other signaling components, such as MAP (mitogen-activated protein) kinases. To achieve this goal, the early developmental expression of discoidin (Dsc) in Dictyostelium will be examined in cells lacking the MAP kinase Erk2 and expressing different wild-type or chimeric G (alpha) subunits. In addition, a genetic screen will be used to identify suppressor mutations that rescue pDSC/GFP reporter gene expression from the repression of a chimeric G (alpha) subunit. The suppressor mutations will be created by REMI mutagenesis, allowing genes to be tagged that might function downstream of G (alpha) subunits to regulate gene expression. Additional characterization of suppressor genes will be conducted to verify their function regulating DSC gene expression downstream of G (alpha) subunits in these signaling pathways. These studies will provide significant insights into the signaling mechanisms that control the differentiation of eukaryotic cells. [unreadable] [unreadable] [unreadable]